In the fields of medical, dietary, environmental and chemical sciences there is an increasing need for the selective separation of specific substances from complex mixtures of related substances. The aim can be the quantitative extraction of a certain compound or compounds, the measurement of their concentration or the selective removal of a target compound from a multi-component mixture.
Stricter health controls have increased the demand for methods allowing sensitive and selective quantification of hazardous products and metabolites from certain everyday substances in widespread use. Of particular concern are chemical compounds related to use of tobacco-based products, which compounds are either originally present in the raw tobacco leaf itself or generated during the smoking process. Nitroso-containing compounds, such as nitrosamines, are regarded as being of special significance in this regard.
With the aim of reducing the occurrence of hazards related to smoking, certain pharmaceutical products have been produced containing only the neuroactive substance, nicotine, the chemical claimed to be responsible for the dependence aspects of smokable material.
Among the nicotine formulations for smoking cessation therapy, nicotine chewing gum has found the most widespread use. The quality control required during production includes monitoring of the nicotine level (2 or 4 mg per gum) as well as monitoring of the primary nicotine oxidation products cotinine, myosmine, nicotine-cis-N-oxide, nicotine-trans-N-oxide and beta-nicotyrine. Quantitation of nornicotine, anatabine and anabasine is also desirable, if not required. According to the United States Pharmacopeia (U.S.P.) the gum formulation should contain between 95% and 110% of the amount of nicotine given on the label and the amount of each oxidation product should not exceed 0.1% of the nicotine amount.
Despite the use of such cigarette substitutes, nitrosamine nicotine metabolites may be produced in vivo by natural metabolic processes during the residence of the nicotine within body tissues. The levels of these metabolites remain below the concentrations at which most analytical procedures can perform quantitatively. The need for methods which are capable of monitoring these levels, as well as the levels of other nicotine metabolites, is therefore of importance. Typically, such monitoring is performed on human urine samples in which levels of such suspected carcinogens are extremely low.
Targeted compounds for quantification, reduction or removal from tobacco or smoke are known and include the major components of tobacco-specific nitrosamines (TSNAs) and their alkaloid precursors: NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; NNA, 4-(methylnitrosamino)-4-(3-pyridyl)butanal; NNN, N-nitrosonornicotine; NAB, N-nitrosoanabasine; NAT, N-nitrosoanatabine; NNAL, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol; iso-NNAL, 4-(methylnitrosamino)-4-(3-pyridyl)-1-butanol; iso-5 NNAC, 4-(methylnitrosamino)-4-(3-pyridyl)butanoic acid.
To properly quantify how much of such targeted compounds are present in human biological fluids, methods are being developed to analyse the alkaloids, especially the nitrosylated decomposition products and metabolites in tobacco. Existing chromatographic separation or extraction methods used for this analysis lack the robustness, sensitivity and speed required in order to handle the large number of samples generated when screening the general population. With existing methods, the low concentration of the nitrosamines, which are typically present in picograms per milliliter, demands extensive sample preparation with multi-step extractions and often chemical derivatization (for example deuteration prior to mass spectrometry) of the analyte prior to analysis. One reason for this complexity is that the existing separation materials are not selective as, for example, an antibody or biological receptor might be for the metabolites in question but rather rely on physico-chemical properties like charge or hydrophobicity of the metabolites for the separation behaviour. These physico-chemical properties may be shared by many other irrelevant molecules in the sample.
A typical procedure might involve up to seven work-up steps including centrifugations, pH adjustments, enzymatic treatments, etc., which may sum up to a preparation time of many hours or even days per sample. With such cumbersome procedures, loss of material during the process can lead to errors in estimation of the original sample concentrations, requiring extrapolation back from the final measurement, rather then reliance on direct measurement, to obtain the original concentration in the sample. A quick and simple method for the analysis of tobacco-specific nitrosamines is therefore a significant unmet medical analytical need. (See, e.g. Byrd & Ogden, Journal of Mass Spectrometry, 2003, 38, 98-107 and Wu et al. Anal. Chem. 2003, 75, 4827-4832).
During recent years numerous reports of selective recognition of small molecules with materials prepared by molecular imprinting (molecularly imprinted polymers or MIPs) have appeared. See, for example, Wulff, G. Angew, Chemie. Int. Ed. Engl. 1995 (34) 1812. MIPs are polymers having reactive sites adapted to bind selectively with targeted compounds. Non-covalently prepared molecularly imprinted materials have been used for chiral recognition of a variety of small molecules including therapeutic drugs, sugars, nucleotide bases, and pesticides as well as steroid and peptide hormones. Examples of the same are described in, for example, Sellergren, B. Trends Anal. Chem. 1997 (16) 310. The high affinity and selectivity for the target analyte exhibited by some of the imprinted materials have justified a comparison with the corresponding immuno-affinity (IA) phases. In contrast to the latter phases however, the MIP materials are straightforward to prepare, stable in most media and reusable over long periods of time. Applications of the MIP materials in chromatography, separation (continuous or batch), chemical sensing or in specific assays are therefore under investigation.
Another application is solid-phase extraction (SPE, see Mayes, A. G.; Mosbach, K. Trends Anal. Chem. 1997, 16, 321) of analytes present in low concentrations in biological samples, or in complex matrices. SPE may lead to selective enrichment and clean-up of an analyte to levels not achievable with existing methods. Molecularly imprinted solid phase extractions (MISPE) have been used in bioanalysis, food analysis and environmental analysis. In these examples selective enrichment and clean-up of the analyte is obtained resulting in higher accuracy and a lowering of the detection limit (LOD) in the subsequent chromatographic (eg HPLC) or mass spectrometric quantification.
In view of their high selectivity combined with good affinity for the target molecule or a group of target molecules, MIPs have attracted considerable interest from the food industry as a tool to improve food quality. This requires the use of a MIP for selective removal of undesirable components from the food matrix. Since these components are often present in low concentrations, the saturation capacity of the MIP is typically not a limiting factor.
The preferred specifically designed MIP material of the invention is capable of selectively absorbing the most common nitrosylated nicotine derivatives from complex matrices, such as urine, giving quantitative recovery and thereby leading to low errors in the estimation of such hazardous chemical concentrations.
In addition to quantification it is also well known to attempt to reduce the harmful effects of consuming material containing tobacco, tobacco substitutes or mixtures thereof by reducing the levels of targeted compounds. Such reductions can be made in the material itself or in a derivative thereof such as an extract of the material. Reduction can also be effected in the thermal decomposition products of the material, i.e. mainstream and sidestream smoke obtained by combustion, or the aerosols produced by heating the material to a temperature below its combustion temperature.
One very well known method for this sort of reduction is to contact the thermal decomposition products of the material with a filter that adsorbs undesired components therefrom. An alternative method involves solvent extraction of the material, for example as disclosed in the specification of U.S. Pat. No. 5,601,097. According to that specification, 5 the protein content of tobacco material is reduced by treating the tobacco with a solution containing a surfactant to extract polypeptides, separating the solution, removing the surfactant and the polypeptides from the solution, and recombining the solution with the tobacco material. International patent application specification WO 01/65954 discloses a process in which tobacco is contacted with a supercritical extraction fluid such as supercritical carbon dioxide to selectively reduce or eliminate nitrosamines.
These processes are equally applicable to both tobacco itself and to tobacco substitutes i.e. natural or synthetic materials having similar characteristics to natural tobacco that enable them to be consumed in a similar way to tobacco, whether by smoking, chewing, inhalation or otherwise.
There has been an attempt to remove nicotine from tobacco smoke using MIPs, as reported in Liu, Y., et al., Molecularly imprinted Solid-Phase Extraction Sorbent for Removal of Nicotine from Tobacco Smoke, Analytical Letters, Vol. 36, No. 8, pp 1631-1645 (2003). The MIP described in the article was designed to bind nicotine and not the more toxic nicotine metabolites such as nitrosamines. It is unclear if the MIP was in fact selective for nicotine as the scientific method producing the data was lacking in key control-checking elements.
Therefore, there remains a need in the art for novel MIPs and methods of employing the same, particularly in the field of nicotine and nicotine metabolites.